Byington was born in Colorado Springs, Colorado, the daughter of Edwin Lee Byington, an educator and superintendent of schools in Colorado, and his wife Helene Maud (Cleghorn) Byington. She had a younger sister, Helene Kimball Byington. Her father died in 1891, and her mother sent her younger daughter to live with her grandparents in Port Hope, Ontario, while Spring remained with relatives in Denver. Helene Byington moved to Boston and enrolled in the Boston University School of Medicine, where she graduated in 1896. She then returned to Denver and opened a practice with her classmate, Dr. Mary Ford.

Byington performed occasionally in amateur shows as a student, graduating from North High School in 1904. She soon became a professional actress with the Elitch Garden Stock Company.[2] When their mother died in 1907, Byington and Helene were legally adopted by their aunt Margaret Eddy. Byington stated in a 1949 interview that she briefly tried newspaper reporting. However, since she was already of legal age, she decided to start her acting career in New York City, saying that she enjoyed it, and, "I can't do anything else very well."[3]

In 1908, Byington joined a repertory company that was touring Buenos Aires, Argentina. Between 1908 and 1916, the company performed American plays, translated into Spanish and Portuguese in Argentina and Brazil.

Upon returning to New York, Byington divided her time between working in Manhattan and staying with her daughters. Her daughters were living with friends J. Allen and Lois Babcock, in Leonardsville Village, New York, who were taking care of them while Byington worked in the city. She began touring in 1919 with a production of Birds in Paradise, which brought the Hawaiian culture to the mainland, and, in 1921, began work with the Stuart Walker Company, for which she played roles in Mr. Pim Passes By, The Ruined Lady, and Rollo's Wild Oats, among others. This connection landed her a role in her first Broadway performance in 1924, George S. Kaufman and Marc Connelly'sBeggar on Horseback which ran for six months. She renewed the role in March and April 1925, and continued on Broadway with an additional 18 productions in the ten years from 1925 to 1935. These included roles in Kaufman and Moss Hart'sOnce in a Lifetime, Rachel Crothers's When Ladies Meet, and Dawn Powell'sJig Saw.

During World War II, Byington worked in radio, and decided to continue working in this medium, as her film career began to decline after the war. In 1952, she joined CBS Radio to become the lead role of the widowed Lily Ruskin, in the sitcomDecember Bride. In 1954, the television company Desilu Productions produced a pilot of the show for a sitcom, also starring Byington. The pilot was successful, and the new hit sitcom aired in its first two seasons immediately following I Love Lucy. December Bride broadcast 111 episodes through 1959.

From 1961 to 1963, Byington was cast as the wise, matronly housekeeper, Daisy Cooper, in the NBCWestern series Laramie, starring John Smith and Robert Fuller. On Laramie, Daisy serves as a surrogate grandmother to orphaned Mike Williams, played by the child actor Dennis Holmes.

Byington spoke some Spanish, which she learned during the time spent with her husband in Buenos Aires; and she studied Brazilian Portuguese in her golden years. In July 1958, she confided to reporter Hazel Johnson that she had acquired a "small coffee plantation" in Brazil the month before and was learning Portuguese. "Miss Byington explained that she first listens to a 'conditioning record' before she goes to sleep. An hour later, her Portuguese lessons automatically begin feeding into her pillow by means of a small speaker."[2]

In 1909, Spring Byington married Roy Chandler, the manager of the theater troupe with which she worked in Buenos Aires. They remained there until 1916, when Spring returned to New York to give birth to her first daughter, Phyllis Helene. Her second daughter, Lois Irene, was born in 1917. The couple divorced about 1920. Between then and the mid-1930s, she devoted her time to developing her career.[6]

In the late 1930s, Byington was once again engaged to be married, this time to an Argentine industrialist. Following an engagement of a few years and several months before they were to be married, he died unexpectedly. Following this, she chose to devote her life to her career and family.

A number of Hollywood historians have claimed that Byington was a lesbian.[7][8][9][10] Actress Marjorie Main's biographer Michelle Vogel has noted that Main and Byington were reported widely as having had a long-term relationship.[11] When asked about Byington's sexual orientation, Main acknowledged: "It's true, she didn't have much use for men."[7]